The long term objective of this work is to understand how a eukaryotic chromosome replicates. We have prepared a replication map of chromosome III of the yeast, Saccharomyces cerevisiae. This map includes the positions of ARS elements, detected by their ability to promote autonomous replication of plasmids, the positions of chromosomal replication origins, detected by the analysis of replication intermediates, and the positions of DNA replication termination. This proposal addresses three major issues concerning the replication and maintenance of chromosome III. First, both genetic and molecular approaches will be used to study the mechanisms by which a derivative of this chromosome lacking replication origins is maintained. The primary focus of this aim is to identify the mutated genes that cause defects in the maintenance of this 'originless' fragment. Second, we will study the role of EBS1 in maintaining the 'originless' fragment. A dominant, gain-of-function mutation in EBS1 causes both defects in fragment maintenance and UV sensitivity. Finally, we will explore four aspects of replicator structure and activity to test and refine our understanding of replicator structure and the regulation of replicator activity. In this aim, we will study how multiple initiator binding sites contribute to the activity of a compound replicator; study the role of Ctf7p, which is required for the establishment of sister chromatid cohesion, in replication initiation; study the role of ARS304 in the activity of the recombination enhancer, which controls donor selection in mating type switching; and study the basis for differences in replicator activity observed in two yeast strains. These studies will provide new insights into a fundamental cellular process, chromosome replication. Knowledge gained from the genetically tractable yeast system is applicable to larger eukaryotes, and guides approaches to these experimentally difficult systems. The proposed studies are also likely to lead to a further understanding of the molecular basis of genome stability.